EdwardRyanTalatala Week 3
Purpose
The purpose of this assignment was to learn how to properly read primary research articles, i.e. how to read the paper critically and how to criticize and review the paper. It also teaches us how to interpret figures in the article, as well as how to present them in open discussion in class.
Journal Club 1 Assignment
Biological Terms
- Ubiquitin: a small protein used for regulation in eukaryotes (Biology Online, 2006)
- Shine-Dalgarno sequence: a small sequence of nucleotides upstream the translational start site of a prokaryotic mRNA molecule, that assists in binding of rRNA and bringing initiator codon to the mRNA. (Biology Online, 2005)
- dimorphic: of two different forms (in this case of fungi: mold & yeast) (Biology Online, 2005)
- trehalose: a disaccharide made from two alpha-glucose molecules, which functions as an energy source for some bacteria, fungi, plants, and invertebrates (Biology Online, 2016)
- Dendrogram- a branching diagram representing a hierarchy of categories based on degree of similarity or number of shared characteristics especially in biological taxonomy (Merriam-Webster, 2019)
- glutathione transferase: A family of enzymes involved in metabolism and in making toxic compounds less harmful to the body (National Cancer Institute, 2019)
- isoforms: Any of the proteins with the same function and similar amino acid sequence, encoded by different genes or by RNA transcript (Biology Online, 2009)
- glutaredoxin: A protein involved in the reaction of mitochondrial redox signal and oxidative stress through facilitating the interchange of the mitochondrial glutathione pool and protein thiols (Biology Online, 20014)
- thioredoxin peroxidase: An enzyme upholds thioredoxin in reduced state that involved in the defense against oxidative stress and redox-regulatory mechanism oftranscription factors to various biological phenomena. (Biology Online, 2014)
- desaturase: Any of several enzymes that putdouble bonds into the hydrocarbon areasof fatty acids.(Biology Online, 2005)
Article Outline
What is the main result presented in this paper?
The main result presented in this paper is that S. cerevisiane has two transcriptional expression patterns in response to cold, the early phase and the late phase. The early phase stress response is related to a change in membrane fluidity in response to cold, as well as RNA secondary structures destabilization. The late phase stress response was contributed to environmental stresses that altered the physiology of the cell.
What is the importance or significance of this work?
This paper demonstrated the ability of yeast cells to mitigate stress responses by altering gene expression.
What were the limitations in previous studies that led them to perform this work?
Previous studies have studied the effects of heat shock on yeasts, but not on the effects of cold shock. This led to researchers to conduct this study to determine the effects of cold shock on yeast cells.
How did they treat the yeast cells (what experiment were they doing?)
They induced a variety of stress stimuli, including cold shock, heat shock, and environmental stresses (menadione, oxidative stress, osmotic stress, disulfide reducing agent)
What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
BY4743 and BSY25 wild-type, msn2, and msn4 mutant cells. Diploid.
What media did they grow them in? What temperature? What type of incubator? For how long?
They used YPD medium, which is made of glucose, bactopeptone, and yeast extract, and they were inoculated at 30 degrees for one night.
What controls did they use?
The control microarrays were grown at 30 degrees
How many replicates did they perform per treatment or timepoint?
- Two replicates at 0 and 2 hours. Three replicates for 12 hours.
- Three repeats at 10, 30, and 60 minutes.
What method did they use to prepare the RNA, label it and hybridize it to the microarray?
RNA was isolated using the hot-phenol method, wherein they used acid phenol, vortexing, centrifuging, and incubating cultures at different temperatures. Then it was purified using the Oligotex Spin-Column Protocol. Then it was labeled using reverse transcription of Cy3- and Cy5-dCTP. Finally, it was hybridized onto yeast genomic DNA microarrays.
What mathematical/statistical method did they use to analyze the data?
They used the ScanArray lite scanner to scan the microarray slides. Then they had to pass quality control tests: 1) signal intensity was significantly greater than the background; 2) signal intensity was within the dynamic range of the photomultiplier tube; and 3) Raw intensities of duplicate spots for each gene had to be within 50% of one another
Are the data publicly available for download? From which web site?
http://cbr-rbc.nrc-cnrc.gc.ca/genetics/cold/
Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
Figure 1a
X-axis: a variety of genes Y-axis: the upregulation or downregulation of these genes at different time points Measurements: microarray data and clustering was done using the GeneSpring software Trends/Conclusions: Using this data, the researchers were able to determine the ECR and LCR genes
Figure 1b/c
X-axis: specific gene categories Y-axis: number of genes upregulated or downregulated Measurements: microarray data and clustering was done using the GeneSpring software Trends/Conclusions: Early response upregulates genes mainly concerning transport, transcription, and metabolism (also down-regulates mostly metabolism-related genes) Late response upregulates genes mainly concerning metabolism stress response (downregulates genes concerning metabolism and protein synthesis)
Figure 2
X-axis: different time points and the decrease in temperature Y-axis: color map to show the fold change in ECR genes Measurements: microarray data Trends/Conclusions: The repressed genes in cluster A remained repressed with the decrease in temperature, while 47% of the genes in cluster B increased expression with the decrease in temperature.
Figure 3a
X-axis: time with the different stresses Y-axis: color map to show the fold change in ECR genes Measurements: microarray data after varying time periods, depending on stressor Trends/Conclusions: The clusters labeled I that half of the repressed ECR genes were induced during heat shock, and the cluster labeled II showed that 40 % of induce heat shock genes were repressed.
Figure 3b
X-axis: time with the different stresses Y-axis: color map to show the fold change in LCR genes Measurements: microarray data after varying time periods, depending on stressor Trends/Conclusions: LCR genes show that the genes that were reciprocated during ECR returned to their normal expression over time
Figure 3c
Trends/Conclusions: These Venn diagrams compares the genes that were induced or repressed in ECR, LCR, and ESR. It compared which genes reciprocated with stress, as well as which genes demonstrated coexpression or corepression. They concluded that LCR involves ESR, while ECR demonstrates specific responses to cold shock.
Figure 4
X-axis: times at 0, 2, and 12 hours Y-axis: color map demonstrating induced or repressed gene expression Measurements: Microarray data of experiment Trends/Conclusions: Msn2p/Msn4p not significantly affecting the LCR genes reveals that there is a cold specific response in the ECR.
Figure 5
X-axis: varying time periods Y-axis: levels of glycogen and trehalose Measurements: used Sigma-Aldrich kit to determine sugar levels Trends/Conclusions: these observations support microarray data because genes that regulate carbohydrate metabolism were induced after 12 hours of cold.
Figure 6
X-axis: different time periods and the comparisons from this experiment to the Sahara et al. (2002) study Y-axis: transcriptional responses of the varying genes to cold Measurements: compared gene clusters of this experiment to those of the Sahara experiment Trends/Conclusions: Controlling for similar variable, this study found that there were different results for the gene expressions to different stressors, probably from the use of different strains or experimental designs.
How does this work compare with previous studies?
This work effectively filled in gaps for transcriptional responses to cold shock and other environmental stressors for yeast cells, a knowledge that has yet to be tested.
What future directions should the authors take?
The authors should use this newfound information to apply it to humans. Maybe to determine how human genes respond to cold and other environmental stressors to find way to mitigate these changes, potentially allow us to survive in more extreme conditions.
Give a critical evaluation of how well you think the authors supported their conclusions with the data they showed. Are there any major flaws to the paper?
The authors did a great job of compacting a vast amount of data to these figures. The article was very difficult to understand, requiring me to read each paragraph several times very carefully to understand that they were talking about. This is probably just due to my inexperience to yeast research jargon. However, this did help me gain a greater understanding in gene expression and transcriptional responses to stress stimuli. I believe the conclusions were acceptable with the data and with how I understood the article.
Figure 3
Acknowledgments
- I worked with my homework partner Austin Dias a few times outside of class to discuss our figure and to discover the main conclusions of this research paper.
- Previous lecture slides from Dr. Dahlquist and Dr.Fitzpatrick provided the resources necessary to complete this task, such as the links to useful dictionaries.
Except for what is noted above, this individual journal entry was completed by me and not copied from another source. EdwardRyanTalatala (talk) 23:48, 6 February 2019 (PST)
References
Dahlquist, K. and Fitzpatrick, B. (2019). BIOL388/S19:Week 3. [online] openwetware.org. Available at:Week 3 Assignment Page [Accessed 6 Feb. 2019].
Biology Online (2006, June 26) Ubiquitin. Retrieved from https://www.biology-online.org/dictionary/Ubiquitin on 06 February 2019.
Biology Online (2005, Oct 3) Shine-Dalgarno Sequence. Retrieved from https://www.biology-online.org/dictionary/Shine-dalgarno_sequence on 06 February 2019.
Biology Online (2005, Oct 3) Dimorphic. Retrieved from https://www.biology-online.org/dictionary/Dimorphic on 06 February 2019.
Biology Online (2016, Sept16) Trehalose. Retrieved from https://www.biology-online.org/dictionary/Trehalose on 06 February 2019.
Merriam-Webster Online (2019) Dendrogram. Retrieved from https://www.merriam-webster.com/dictionary/dendrogram on 06 February 2019.
National Cancer Institute Online (2019) glutathione S-transferase. Retrieved from https://www.cancer.gov/publications/dictionaries/cancer-terms/def/glutathione-s-transferase on 06 February 2019.
Biology Online (2009, Nov 9) Isoform. Retrieved from https://www.biology-online.org/dictionary/Isoform on 06 February 2019.
Biology Online (2014, Nov 19) Glutaredoxin-2. Retrieved from https://www.biology-online.org/dictionary/Glutaredoxin-2 on 06 February 2019.
Biology Online (2014, Nov 28) Thioredoxin reductase 2. Retrieved from https://www.biology-online.org/dictionary/Thioredoxin_reductase_2 on 06 February 2019
Biology Online (2005, Oct 3) Desaturase. Retrieved from https://www.biology-online.org/dictionary/Desaturase on 06 February 2019.
